Abstract
To elucidate the dynamics of oxidative stress in fish, it is necessary to know the concentration of superoxide anions as a precursor to various reactive oxygen species in the living body. Superoxides are unstable radicals, disappearing within a few seconds, which makes it challenging to measure them using conventional methods. We describe herein the development of a biosensor that can rapidly quantify superoxide concentrations in biological samples. The sensor was fabricated by immobilizing cytochrome c as an electron transfer element on the surface of a gold electrode as a self-assembled monolayer. The fabricated sensor was immersed in hypoxanthine solution as a working electrode with an Ag/AgCl counterelectrode. Xanthine oxidase standard solution at different concentrations was then added to generate superoxides. The sensor output correlated well with the superoxide concentration in the range of 0.58–9.33 µM. To confirm the specificity, various substances that may affect the sensor response were examined, revealing little effect on the sensor output. The sensor was applied to eyeball interstitial sclera fluid of Nile tilapia Oreochromis niloticus and exhibited acceptable performance.